All mice were housed in standard cages in a temperature controlled (22 ± 2°C) and pathogen-free room under diurnal conditions (12 hour light/dark cycle) with food and water available ad libitum. All animal procedures were in accordance with the guidelines on the Care and Use of Animals for Scientific Purposes developed by the National Advisory Committee for Laboratory Animal Research (NACLAR) and approved by Institutional Animal Care and Use Committee (IACUC) of the National Neuroscience Institute.
Transgenic mouse production
A targeting vector was designed to contain the VPS35 D620N mutation gene downstream of a loxP-flanked stop sequence (LSL). C57BL/6J-LSL-VPS35 D620N/- mice were subsequently generated by microinjecting this entire construct, which was inserted into the ROSA26 locus, into the pronuclei fertilized oocytes of pseudopregnant C57BL/6j females. After establishing germ-line transmission, two founder lines were first backcrossed to produce homozygous founders (C57BL/6J-LSL-VPS35 D620N/D620N).
The homozygous founder line (C57BL/6J-LSL-VPS35 D620N/D620N) bearing the VPS35 D620N allele can only be expressed after Cre-mediated excision of the STOP cassette. Hence, the homozygous founder lines were crossed with C57BL/6J mice (National University of Singapore Comparative Medicine Centre) first to obtain hemizygous mice (C57BL/6J-LSL-VPS35 D620N/-). Then, the hemizygous mice were crossed with the C57BL/6J-Nestin-cre mouse strain purchased from Jackson Laboratories. Progeny, including C57BL/6J-VPS35 D620N/- (designed as the Tg mice) and C57BL/6J-VPS35 -/- (designed as the NTg mice) mice, were genotyped for the presence of the VPS35 D620N allele using specific PCR primers (VPS35s5: 5’-TTGCGCTTGCGGCGGGAATCACCAG-3’ and VPS35gt1: 5’-ACACTGGAGCATTTGCGCTTGCGGC-3’). Similarly, sense primers (5’- CCGGTGAACGTGCAAAACAGGCTCTA-3’) and anti-sense primers (5’-GATTAACATTCTCCCACCGTCAGT-3’) were used to detect the presence of the Nestin cre gene sequence.
Neuropathological characterization of transgenic mice
VPS35 D620N Tg mice and age-matched control NTg mice were anesthetized with ketamine/xylazine (100/10 mixture; 0.1 mg g−1 body weight, intraperitoneal injection) and perfused with ice-cold PBS followed by 4% paraformaldehyde in PBS. Mouse brains and selected organs were collected, dissected and soaked in fixative for 24 hours and then transferred into 30% sucrose buffer for 2-3 days until they sank down to the bottom of the tube. Subsequently, brain samples were sliced into 30 µm coronal section series on a Micromcryostat (Leica CM3050S) at −20°C. Immunofluorescence staining was performed on free-floating sections that were then stained in a solution containing 0.1% BSA, 0.1% Triton X-100 and the rabbit Anti-HA tag [HA. C5] antibody (Abcam#ab18181), and rabbit anti-NeuN antibody (Millipore#ABN78). After a series of 0.1M PBS washes, sections were stained using the same blocking solution as above and Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 555 donkey anti-mouse secondary antibodies (Inventrogen).
Immunohistochemistry (IHC) staining
35 µm coronal brain sections of NTg and Tg mice were washed with Dulbecco’s phosphate-buffered saline (PBS) pH 7.4 for 5 minutes. They were blocked in 1% BSA including 0.1% Triton X-100 at room temperature for 30 minutes, which was followed by incubation of the primary antibodies overnight at 4OC. The following primary antibodies were used in this study (1:200) : rabbit anti-HA (Cell signaling technology, #3724S) and mouse anti-TH (Millipore, #MAB318). Next, brain sections were washed with PBS 3 times, for 5 minutes each time, and incubated with secondary antibodies (1:400) : goat anti-mouse Alexa Fluor 488 (Invitrogen, #A-11001) and goat anti-rabbit Alexa Fluor 555 (Invitrogen, #A-21428). Brain sections were also counterstained with the fluorescent nuclear dye 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, #B2261). Subsequently, brain sections were washed with PBS again (3 times, 5 minutes each time). Finally, brain sections were washed, dried and mounted with DAKO Fluorescence Mounting Medium.
Stereology cell counting
Using the previously collected mouse brain samples, 35 µm coronal sections were cut (Leica CM1950), and free floating sections were incubated overnight at room temperature with rabbit anti-TH primary antibody (Novus Cat. NB300-109), followed by incubation for 1 hour with secondary antibody using the Elite rabbit IgG kit (vector laboratory PK-6101) (41). The DAB kit (Vector laboratory PK-4100) was used for color development.
The number of TH positive dopamine neurons in the SN was estimated using unbiased stereological counting. The SN was delineated from 1.70 to 3.88 mm posterior to bregma using the Allen brain atlas (42). A total of 7 coronal sections from the midbrain of each mouse were used for quantification (1 section from every 5 serial coronal sections across the midbrain was used); representative pictures containing TH+ neurons in the SN were taken using the 10x magnification objective lens of an Olympus IX83 microscope. Scale bar, 500 µm. The number of TH+ neurons in each section was stereologically quantified using Stereologer 2000TM, at 63x magnification. The 7 sections from each mouse were then used to compare the number of TH+ neurons in Tg and NTg mice (n=4 for Tg, n=3 for NTg).
HPLC measurement of DA, DOPAC and HVA
The brains of VPS35 D620 transgenic and age-matched NTg mice (n=6 for NTg, n=4 for Tg) were quickly removed and washed with ice-cold phosphate-buffered saline (PBS). The striatum of the right hemispheres were removed. Mice striatal tissue was lysed in 0.5M perchloric acid. The levels of DA, DOPAC and HVA of the tissue were measured by Reversed-phase UltiMate 3000 HPLC system (Germany) with ECD detector and a reversed-phase column (DBS HYPERSIL C18, 25cm x 3.0mm, 5 μm particle size, 130 A° pore size) and analysed under the control of Chromeleon™ 7.2 Chromatography Data System. The mobile phase was a mixture of 1.3% NaAc, 0.5% sodium 1-heptanesulfonate, 0.01% EDTA (adjusted to pH 4.0 with 100% acetic acid) and 2% methanol (v/v), 7% Acetonitrile (v/v). All solutions for HPLC analysis were double filtered through 0.2 μm membranes and degassed before use. The flow rate was 1 ml per minute. All chemicals were purchased from Sigma-Aldrich.
Protein extraction and western blotting
Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0°C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500μl, 5μl aprotinin, PMSF 50μl, Na3VO4 100μl and NaF 20μl in 4.32 ml PBS. Extracts were clarified by centrifugation at 4°C (13,200 g for 20 minutes). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE (43). The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), a-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.
ARG evaluation of [3H]FE-PE2I binding
In vitro ARG evaluation was performed on striatal sections. NTg (n=3) and Tg (n=3) mice were sacrificed by CO2, and mice brains were freshly dissected and frozen in -80°C. 10µm thick brain sections containing the dorsal part of striatum were cut and thaw-mounted on Superfrost Plus glass slides then stored at -80°C until ARG was performed.
ARG of the dopamine transporter was carried out using [3H]FE-PE2I as a radioligand. Sections were pre-incubated for 15 minutes in 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2mM CaCl2 and 1 mM MgCl2. Incubations were carried out for 1 hour in 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 1 nM [3H]FE-PE2I (specific radioactivity, 39 Ci/mmol). Non-specific binding was determined in adjacent sections in the presence of 10 μM GBR12909. Sections were washed 3 times (3 minutes for each wash) in cold (4 ºC) 50 mM Tris-HCl buffer, pH 7.4, followed by a brief dip in distilled water. Radioactivity was detected and quantified using a phosphor imager (scanner: Fuji BAS-5000 image reader; imaging plates: BAS-TR2025, Fujifilm, Tokyo, Japan). The measured photostimulated luminescence (PSL) /mm2 values were transformed into radioactivity units and into binding density (pmol/g tissue) based on intensity values obtained using tritium standards (Microscales, American Radiolabeled Chemicals Inc.). Regional specific binding was calculated by subtracting nonspecific binding, as defined in the presence of 10 μM GBR12909, from the total [3H]FE-PE2I binding.
Heterozygous VPS35 D620N mice were crossed with homozygous Nestin cre mice which gives rise to progeny that are 50% Tg and 50% NTg for VPS35 D620N, with both groups of mice carrying the Nestin cre gene. Genotype of the animals was determined by performing PCR on tail samples. Mice were marked randomly at the ears and were tested in all experiments in the same order according to their identification number (number/cage). The experiments and analysis were always performed blind of the genotype. All efforts were made to minimize suffering of the 20-months old Tg and NTg mice. Only one test was performed per day, ranging from 9∶00 AM to 5∶00 PM. On the testing day, animals were put in the test room at least 20 minutes before testing in order to acclimatize. The entire behavior assay took 2 months. The details of the behavioral tests are described below:
Mice were allowed to explore freely in an arena of 50 x 50 x 50 cm for 15 minutes. Each mouse was gently placed in the middle of cleaned grey color perspex chamber at the start of the test session, was allowed to explore the arena undisturbed for 15 minutes, and then removed. The arena was cleaned with 70% ethanol between animals. Video analysis and data acquisition were performed with ANY-mazeTM video tracking system (Stoelting, USA) to analyse total distance, total movement duration, moving speed, average speed, and duration at the periphery and center zone.
Mice were placed on an accelerating rotarod for a maximum of 5 minutes (4 to 40 rpm, 5 minute ramp, UgoBasile, Italy). The latency to fall from the rotating rod was taken over 3 days, with 2 trials per day and an inter-trial period of 2 hours. The last 2 trials of the 3rd day served as a mean value for locomotor abilities. Immediately after each session, the apparatus was cleaned with 70% ethanol.
Elevated plus maze
The 53 cm high grey colour high-tech metal alloy maze apparatus, consisted of two open arms (80 x 5 cm) and two closed arms (80 x 5 cm; surrounded by 15-cm-high walls) arranged in a plus shape. The central platform (5 x 5 cm) served as the convergence site of the four arms. A video camera was attached above the set-up to automatically record each trial. Mice were gently placed on the central platform facing a closed arm and were allowed to freely explore the maze for 5 minutes. The apparatus was cleaned immediately after each session with cotton pads wetted with 70% ethanol. The test was automatically analysed with the ANY-mazeTM videotracking system (Stoelting, USA).
Tail suspension test
Mice were individually suspended by the tail to a horizontal wooden bar 40 cm above the bench top using an adhesive tape placed approximately 1 cm from the tip of the tail. Typically, mice demonstrate several escape-oriented behaviors interspersed with temporally increasing bouts of immobility. The behaviors were videotaped throughout the 6-minute test and the immobility time, defined as lack of all movement except for whisker movement and respiration, was measured with a stopwatch.
Morris water maze
Morris water maze was used to measure the ability of spatial learning and memory. In brief, the water maze was a circular tank (120 cm in diameter and 60 cm in height) located in a room containing a variety of cues. The pool was filled with opaque water that was made by adding white colored non-toxic paint and the temperature was maintained at 25±1 ◦C. The tank was divided into four quadrants and an invisible platform was placed 1 cm below the surface of the water. There were 3 trials per day for each mouse during the 4 days of training. For each trial, the mouse was randomly put into the water in one of the quadrants with its head facing the tank wall. Each mouse was allowed to swim freely until it discovered, climbed onto the hidden platform, and stayed there for 10 seconds. In the event a mouse did not reach the platform after 60 seconds of swimming, it was gently hand-guided to the platform. 24 hours after the 4th day of training, the probe test was performed. Here, the platform was removed and the mouse was subsequently allowed to swim for 60 seconds. The escape latency, the time spent in the target quadrant and the distance traversed in order to find the platform were used to evaluate spatial learning and memory. All trials were processed by a video track tracking system (ANY-mazeTM video tracking system (Stoelting, USA).
The behaviors of the mice were analysed according to the test performed: the activity was automatically recorded in the open field, elevated plus maze and water maze by the ANY-Maze video tracking system (Stoelting, USA), or displayed automatically on the rotarod apparatus (UgoBasile, Italy). Data in the tail suspension test and forced swimming test were collected manually with a stopwatch. Statistics were performed using SPSS Software. Two kinds of analysis were usually done: (i) Analysis of variances by two-way ANOVA followed by post-hoc comparisons (Bonferroni), or the unpaired Student’s t-test for unequal variance. Equality of variances was assessed by Levene’s Test. Significance level was set at *P < 0.05.